我们感谢加斯帕Kronhamn和丹Hultmark，迈克尔Galko酒店和布卢明顿股票中心飞股票。特别感谢康特尼 - 小野寺咨询与统计分析。我们感谢卡特里娜黄金手稿的批判性阅读和卡尔帕纳·麦克贾尼，卡特里娜黄金的Derynck实验室的成员，以及Nystul实验室进行讨论和评论的手稿的成员。这项工作是由加州大学旧金山分校计划赠款突破生物医学研究（PBBR），远大中心，赫尔曼基金会，美国癌症协会RSG DDC-122595，美国心脏协会13BGIA13730001，美国国家科学基金会1326268，卫生1R01GM112083-01全国学院和支持1R56HL118726-01A1（以KB）。

1，血细胞出血/刮分析<ol><li>准备幻灯片: <ol><li>选项​​1的显微镜没有瓦片扫描功能:对于每一个幼虫要分析，制备一种玻璃载玻片用各2毫米的正方形约5巴氏笔井，对应于显微镜的领域观察区域;添加约5 - 10微升S2媒体的每个（图1A）。保持滑动保湿室，以防止井干燥。 </li><li>选项​​2为显微镜用瓷砖扫描功能:对于每一个幼虫待分析，准备为〜3 3至4巴氏笔井1载玻片 - 每4平方毫米;加约15 - 20微升S2介质到每个孔中（图1B）。保持滑动保湿室，以防止井干燥。 注:孔的上述建议的数量是足够的每幼虫总数高达3000血细胞（后第二龄幼虫，〜2.5 - 3毫米的长度，转基因标签的大部分larv的人血细胞）。当评估较大血细胞数目，更多的井可能需要避免过度拥挤。 </li></ol></li><li>收集幼虫: <ol><li>喷射水成含有幼虫和冲洗幼虫到培养皿蝇小瓶，或舀一些食品包含幼虫到培养皿，并用喷瓶，用水稀释。 </li><li>轻轻一挑出来的幼虫用画笔培养皿中，并放置在水中一腔的菜，或在冷块上的幻灯片。 注:幼虫可以保持在水或冷块上的有限的时间;使用在45分以下的标本，以免幼虫死亡或血细胞有害的影响。 </li></ol></li><li>解剖: <ol><li>冷金属块在荧光显微镜下选择幼虫。如果需要测量尺寸和图像幼虫。 </li><li>隔离循环血细胞（"渗透"）的: <ol><li>一旦幼虫被选择时，将一只幼虫在第一巴氏笔井（图1C，2A）。 </li><li>使用2清洁针具或解剖剪刀和镊子，使切口同时在后部和前幼虫的末端。为了避免干扰居民的血细胞，最好是在幼虫腹侧使这些切口。对于一致的结果，使每一个幼虫相同位置的切口。为1日龄幼虫，1切口（在腹侧前）就足够了。 </li><li>允许幼虫流血了几秒钟，没有任何压力或身体搅拌（ 图2A）。 注:如果处理多个幼虫，最好在进行下一步骤，以避免保持幼虫在冰上太长可能影响样品"完整性之前，使这些切口为每一个。 </li><li>轻轻抬起用针或镊子幼虫蘸到第二口井，以冲洗任何剩余的循环血细胞。在此之后，按照与居民血细胞释放。 </li></ol></li><l我&gt;居民血细胞分离（"刮"）: <ol><li>幼虫轻轻转移到下一个孔（图2C）。 </li><li>识别幼虫，其典型地位于约1/3从幼虫的前端的淋巴腺，并且其可以通过幼虫体壁背侧荧光。避免淋巴腺，同时通过牵制的幼虫用针尽可能接近到淋巴腺，以避免刺穿（ 图2C），释放居民的血细胞。 注:在正常发展的淋巴腺血细胞的成熟相比，幼虫的血细胞被延迟，并有区别的血细胞的荧光记者可能不会显示在低龄幼虫的淋巴腺的信号。在这些情况下，关注较少需要由淋巴腺血细胞支付给淋巴腺因为没有污染分化荧光标记的幼虫血细胞的预期。 </li><li>在dissecti释放居民的血细胞拼抢和/或猛刺的过程。使用一个针尖根据需要有效地牵制附近的淋巴腺幼虫（见上文）或其他身体部位。使用其他针戳在血细胞可见的通过幼虫体壁（ 图2C，E）的集群，目标是分开的血细胞。血细胞也可以被释放在一个刮运动。然而，早期的撕裂表皮可能释放出的血细胞，这可能使自动计数更具挑战性的大型集群。 注意:根据幼虫的年龄和基因型，总血细胞的数量将有所不同。分发上述过几口井，以避免一些井血细胞，这可能使单细胞图像分析的难度拥挤的释放过程。 </li><li>如果几个血细胞留在最后胴体，算上这些血细胞通过观察通过手动式计数器（ 图2E）的显微镜和使用。为了方便计算，P花边胎体上相同幻灯片的洁净区和传播它尽可能地薄，以减少光学面的数目。 </li><li>一旦解剖完成后，等待之间5 - 10分钟使细胞成像孔之前沉淀（但不一定附着）。孵育幻灯片的保湿室，以避免干燥，避免野蛮装卸的幻灯片，这可能会扰乱安定血细胞。 注:当确定血细胞数量，释放的细胞是不固定的，细胞必须被成像解剖后不久，优选在从幼虫释放后30分钟。 10分钟，这应该通过聚焦通过在阱的介质的光学平面的确认 - 视的介质和电池特性在卷上，将细胞中的绝大多数将已在5结算。但是，血细胞只有一小部分将已经粘附至所述滑动表面通过这个时候，也需要考虑的事实，如果修改这一协议，进行细胞固定基approache秒。 </li></ol></li></ol></li><li>定量: <ol><li>就拿落户血细胞图像的荧光显微镜（ 图2B，D，F）下。按照与使用ImageJ软件血细胞的定量。 </li><li>准备图像ImageJ的细胞计数算法: <ol><li>采用国内的ImageJ打开图像:文件→打开→（找到文件，并选择）。 </li><li>确保图像（S）是8位或16位。调整阈值的图像通过选择图片 ，然后单击调整 ，然后选择阈值 。遵守" 阈值窗口 "（ 图3A）。 </li><li>检查"黑暗背景"选项。选择"红色"，并增加了低阀值 （见黑色箭头），直到图像中的每个细胞都标有一个红点（即不被覆盖将灰阶可以看到细胞; 图3B）。作为下限阈值增加某些细胞会变得无标记。这对于多远设置下限阈值指示器。 注:有时细胞簇不能得到解决，将被计为一个由粒子计数器。在这种情况下，细胞在一个簇的数目可以通过检查图像（如果需要放大），并使用式计数器人工计数来估计。或者，所述下限阈值可以提高到解决细胞簇;从该操作产生的任何未标记的细胞然后可以使用一式计数器计数。 </li></ol></li><li>使用ImageJ分析细胞数: <ol><li>发射粒子分析仪计数细胞 （图3C）。选择分析 ，然后单击分析粒子。（可选）选择"叠加纲要 "中看到的粒子计数算法（图3D）。可替代地，设置限制向一个单元的大小或像素的区域（ 例如。，细胞， 细胞等）的算法的丛计数。 </li><li>单击确定。观察与计数（ 图3E）的摘要窗口。 </li></ol></li></ol></li></ol> 2.血细胞干扰试验<ol><li>打扰血细胞，选择幼虫，并将其放置在一个2 ml离心管约为0.5克玻璃珠（212 - 600微米），并加入0.5毫升的水。 </li><li>涡管，通过手，以速度10℃1分钟。 </li><li>由溢出的微量离心管中的内容到培养皿并挑选出幼虫用画笔检索来自玻璃珠的幼虫。 </li><li>为恢复阶段，放置在幼虫预先制备培养皿与少量飞食品。允许幼虫以重新建立它们的血球图案的一段45分钟或根据需要。 注:放弃已经停止运动，因为他们在这个过程中已经死亡的任何幼虫。然而，我们通常见升后ittle损害涡旋1分钟（见下文和补充图1）。 </li><li>恢复期后，继续出血/刮含量，如第1如上所述。 </li></ol>

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The authors have nothing to disclose.

这里，我们描述定量从单个果蝇幼虫回收居民和循环血细胞，并量化这两个血细胞种群的第一个方法。该协议包括下列顺序释放循环和驻地的血细胞中，接着成像和自动细胞计数。幼虫居民血细胞可瞬时动员到循环由机械干扰，已知能够在很大程度上30内反转的处理- 60分钟恢复期6。因此，该协议以两种方式进行测试，（1）通过评估每幼虫和循环血细胞过度幼虫发育的过程中的分数的总血细胞数，和（2）通过使用自动化方法实验撞出驻地血细胞，这证实了血细胞定位和驻地血细胞数量和循环群体紧密的联系。另外，该方法的再现性所表明的共mparing生物学重复的两个数据集。 在过去，实验室已使用一系列的技术来量化幼虫血细胞6,13,21。该协议确立了一个共同的标准来检索和果蝇幼虫量化居民和循环血细胞群，提供了一个容易适应的平台。所描述的方法是用于研究，着眼于居民血细胞和其微环境的作用至关重要，造血口袋4-6，并适合于研究荧光蛋白基因携带果蝇品系在野生型和转基因的背景。该协议也是相关的研究，着眼于血细胞动员免疫的挑战和伤害，以及遗传或环境引起的信号触发动员居民血细胞或改变总血细胞数（在4审查）之后。应当指出的是，在二过早的情况下fferentiation和从淋巴腺血细胞的释放，区分胚胎/幼虫与淋巴腺谱系可通过使用的荧光报道血细胞的表达模式来限定。 这里介绍的协议依赖于成像活，荧光标记的血细胞。在将来，它可被修改，以允许释放的细胞的检测固定后， 例如，使用免疫细胞化学。在这种情况下，协议可能需要被调整，以确保血液细胞完全粘附，例如通过增加粘附孵育时间，和加入粘合剂滑动涂层，例如伴刀豆球蛋白A.由于该方法允许血细胞的检索和其操作前体内 ，这将有利于广泛发育，细胞生物学和生物化学的研究。在果蝇和所有胚后发育阶段其他无脊椎动物22，suggesti发现居民和循环血细胞NG这种方法，适应将有利于广泛的超越了果蝇幼虫造血系统的研究。

研究中的无脊椎动物模型果蝇驱使先天免疫1的发现，并且已经促进了血液细胞发展的各个方面的理解2-4。 果蝇造血可分为胚胎/幼虫的血细胞谱系，其起源于胚胎和扩大的幼虫，以及淋巴结的血细胞血统4,5。在这里，我们提出了一个协议，侧重于胚胎/幼虫血细胞谱系，这在果蝇幼虫主要由浆细胞（巨噬细胞）和少量晶体单元4。位于表皮和幼虫体壁5,6的肌肉层之间的幼虫，胚胎血细胞的坚持和殖民节段性重复和终端造血口袋（HPS）。根据其性质，自我更新的巨噬细胞6，局部组织微环境6的主要居住地，从最早的血细胞发育6,8-期间新兴7，和它们的谱系，这血细胞种群被认为类似于脊椎动物自我更新的组织巨噬细胞，一个独立的骨髓系最近在各种物种4,9,10的鉴定。然而，在果蝇中，一些或所有这些常驻细胞也显示可塑性以产生以下的其他血细胞类型如晶单元11,12。 幼虫血细胞主要是驻留（无梗），但在不同的惠普之间的动态稳定状态。它们被逐步释放进入循环，特别是作为第 3龄幼虫接近pupariation 5-7。免疫挑战，受伤或机械干扰导致过早，在后者的情况下是可逆的，动员居民血细胞的成血淋巴4,6,13。 以往的研究表明，居民和循环幼虫血细胞是一脉相承的，但会有不同的粘合剂或归巢性-6,7,13,14。循环与居民血细胞的选择性分离透露增殖水平升高在驻地血细胞的人口，这表明它们暴露于高压钠灯6。 果蝇幼虫惠普感性线索由表皮和肌肉层排列 ​​，并进一步海港周围神经系统的感觉神经元集群（PNS）和肝功能类似oenocytes 6。在功能上，突变和遗传细胞消融实验表明，目前在惠普感觉神经元支持营养存活幼虫血细胞6和定位。 这里，我们描述了具体分离和居民的定量和从单一果蝇幼虫循环血细胞，以及协议为机械血球动员的方法。该方法可用于体外血细胞的研究，并且可以进一步适于诸如蛹和成虫，以及其他无脊椎动物系统其他果蝇发育阶段。由于先前的研究没有居民，循环血细胞区分，该协议规定了居民的血细胞的研究中一个共同的标准，将有助于提高无脊椎动物的血细胞研究的一致性。 首先， 血细胞出血/刮测定描述差分隔离和荧光蛋白标记的居民，循环从单一的果蝇幼虫血细胞种群的自动定量;该协议提供了两种经常和瓷砖扫描配备显微镜（ 图1）。其结果，得到的循环血细胞的百分比和每幼虫血细胞的总数。该方法依赖于表达他们当中的血细胞荧光蛋白转基因果蝇幼虫人口。血球司机或报道的选择将决定结果， 即，该血液细胞群被可视化和量化。主要标记的巨噬细胞（浆细胞），其中包括居民和果蝇幼虫6的循环血细胞人口的绝大多数，合适的转基因包括HMLΔ-红色荧光蛋白6，HmlΔ-GAL4 15，PXN -GAL4 16，CRQ-GAL4（由H. Agaisse 16），或吃-GAL4 17;用于标记的人口相对较少的晶体电池，适合线BcF6-CFP和-GFP 18，或LZ-GAL4（由J.波洛克19）;用于标记lamellocytes，一个专门的细胞类型主要是诱导免疫的挑战和伤害13， 例如，MSNF9mo-mCherry可以使用17。一些转基因的驱动中表达的范围的分化血细胞和祖细胞，如他 - GAL4 20，其中标签约80％的幼虫血细胞20。请注意，在所有的情况下，其中的GAL4的驱动时，与UAS-GFP或另一荧光蛋白UAS -转基因的组合是必需的。在结果部分 ，这种方法用于监测血细胞数量和流通行为在幼虫发育的过程。 第二， 血细胞干扰测定描述旨在通过外部操纵，其随后允许血细胞重新坚持和家庭惠普在有限的时间范围内的能力的评估，以分离驻地血细胞的在先步骤（30 - 60分钟）6。典型地该测定后跟出血/刮除试验，以确定每幼虫循环血细胞的百分比。我们提出了一个简化的协议，用于该测定法（图1D），其使用扰动通过涡旋机智^ h玻璃珠，而不是操纵与油漆刷单幼虫如前面6所述。 在结果部分，该测定是用来证明瞬时分离的血细胞浮在血淋巴，并且可以在循环血细胞的级分中回收。该测定法也是有用的量化血细胞在其归巢/粘附到驻地站 ​​点的差异，比较例如，各种遗传背景或刺激条件。请注意，该机械操作反映了一个可逆的过程，是从infection-或损伤诱发驻地血球动员，这通常是不可逆的，在短时间内帧4,13不同。

<table><tbody><tr><td>6 cm/9 cm Petri dishes</td><td></td><td></td><td>One for each genotype to be evaluated</td></tr><tr><td>Water squirt bottle</td><td></td><td></td><td></td></tr><tr><td>Metal spoon/spatula</td><td></td><td></td><td></td></tr><tr><td>Thin paintbrush</td><td></td><td></td><td>&lt;em&gt;e.g.,&lt;/em&gt; a &quot;liner&quot;</td></tr><tr><td>Glass cavity dish</td><td></td><td></td><td></td></tr><tr><td>PAP pen: Super PAP PEN IM3580</td><td>Beckman Coulter</td><td></td><td></td></tr><tr><td>Glass slides</td><td></td><td></td><td>Each slide will have 5 or more PAP PEN squares drawn on them. Size of squares depends on the imaging objective and magnification of the microscope camera; &lt;em&gt;e.g.,&lt;/em&gt; 2 mm squares.</td></tr><tr><td>Moist chamber</td><td></td><td></td><td>This will be used to prevent slides and wells from drying out: sealed container with wet paper towels lining the sides/bottom</td></tr><tr><td>Schneider&amp;rsquo;s &lt;em&gt;Drosophila&lt;/em&gt; cell culture media</td><td>Invitrogen</td><td></td><td></td></tr><tr><td>Cold block</td><td></td><td></td><td>This is a metal block (a.k.a. heating block) chilled in bucket containing ice; preferably black-colored or other dark, non-reflective color</td></tr><tr><td>2 x 1 ml syringes with needles (27 G &amp;frac12;&quot;)</td><td>Becton Dickinson</td><td></td><td>For dissections.</td></tr><tr><td>Optional: Surgical spring scissors (cutting edge 2 mm)</td><td>Fine Science Tools</td><td></td><td></td></tr><tr><td>Glass beads, 212 - 600 &amp;mu;m</td><td>Sigma</td><td></td><td></td></tr><tr><td>2 ml Eppendorf tubes</td><td>Eppendorf</td><td></td><td>One per genotype evaluating</td></tr><tr><td>Vortex mixer</td><td>Fisher Scientific</td><td></td><td></td></tr><tr><td>Transgenic Drosophila larvae with fluorescently marked hemocytes.</td><td></td><td></td><td>Suitable transgenes include: Hml&amp;Delta;-DsRed (Makhijani &lt;em&gt;et al.,&lt;/em&gt; 2011), MSNF9mo-mCherry (Tokusumi &lt;em&gt;et al.,&lt;/em&gt; 2009), BcF6-CFP and -GFP (Gajewski &lt;em&gt;et al.,&lt;/em&gt; 2007), or Hml&amp;Delta;-GAL4 (Sinenko and Mathey-Prevot, 2004), Pxn-GAL4 (Stramer &lt;em&gt;et al.,&lt;/em&gt; 2005), He-GAL4 (Zettervall &lt;em&gt;et al.,&lt;/em&gt; 2004), Crq-GAL4 (by H. Agaisse (Stramer &lt;em&gt;et al.,&lt;/em&gt; 2005)), or eater-GAL4 (Tokusumi &lt;em&gt;et al.,&lt;/em&gt; 2009) combined with UAS-GFP or other fluorescent protein transgenes.</td></tr><tr><td>Fluorescence dissecting microscope</td><td>Leica</td><td></td><td>Here: Leica M205, optional with camera, imaging software and measuring module</td></tr><tr><td>Inverted fluorescence microscope with camera attachment</td><td>Leica or Keyence</td><td></td><td>With or without tile scanning function (&lt;em&gt;e.g.,&lt;/em&gt; Leica DMI series, Keyence BIOREVO BZ-9000 series)</td></tr></tbody></table>

assaying blood cell populations of the drosophila melanogaster larva

在脊椎动物中，造血由感应微环境（利基市场）监管。同样地，在无脊椎动物模型生物体果蝇 ，称为幼虫造血口袋（HPS）感应的微环境已被确定为解剖学部位的发展和调节的血细胞（血细胞），特别是自我更新的巨噬细胞系的。惠普是分段地重复幼虫的表皮和肌肉层，其还包含外周神经系统的感觉神经元之间的口袋。在幼虫，驻留（无梗）血细胞暴露于抗细胞凋亡，粘合剂和由这些感觉​​神经元增殖性线索和高压钠灯的潜在的其他部件，如内衬肌肉和上皮层。在正常的发展，居民的血细胞逐渐释放从惠普燃料循环血细胞的人口，达到高潮在莫释放ST驻地血细胞在变态的开始。免疫攻击，人身伤害或机械扰动触发过早释放居民的血细胞进入血液循环。居住场所和循环之间幼虫血细胞的开关提出了需要一种通用的标准/过程以选择性地分离和从单一果蝇幼虫量化血细胞的两个群体。因此，本协议描述了一个自动化的方法来释放和量化，从单一的幼虫驻地和循环血细胞。该方法有利于体外方法，以及可适于提供各种果蝇和发育阶段其它无脊椎生物。

为了说明所描述的方法的典型的结果，我们首先使用了血细胞渗/刮测定勾勒幼虫血细胞数量的进展，他们的住所以上幼虫发育的过程中（图4）。居民和循环幼虫血细胞群体是从单一的幼虫中分离（HMLΔ-GAL4，UAS-GFP;他-GAL4标签 使用ImageJ绝大多数幼虫血细胞）和量化。幼虫队列尺寸1.2毫米（〜48小时AEL或1日龄），2.5毫米（〜80小时AEL或2晚第二龄），和3.5毫米（〜96小时AEL或第三龄）进行了检查（图4） 。血细胞数目扩大了幼虫发育的过程中，与相 ​​关并超过以前的估计基于染料染色幼虫7和荧光蛋白标记的血细胞通过幼虫角质层6个现场计数的光学显微镜。在1日插件焦油幼虫几乎所有的血细胞均居于，而循环血细胞的比例逐渐增加了幼虫发育的过程中（ 图4B，C），与以前的出版物6,7一致。 接下来我们检查方法是否忠实地监视居住和流动人口之间血细胞的过渡。以全球化的现象是居民血细胞可以瞬时通过机械扰动脱落，他们重新坚持自己的居住场所自发6，我们通过与作为血细胞干扰测定法中玻璃珠涡旋分散居住血细胞。的确，幼虫机械干扰导致在牺牲居民血细胞（图5）循环血细胞的人口的急剧增加。经过45分钟的恢复期，血细胞已基本回到了自己的附着状态，无论是通过视觉观察，并由作为循环细胞sessed百分比（图5D 中的E）。正如预期的那样，总血细胞数量保持稳定随着时间的推移，尽管血细胞的循环和居民之间的转变。 另外还有几个因素考虑在内。为了证实涡旋没有引起大的组织损伤，与玻璃珠是在台盼蓝（Sigma）的的不同时间（1,5，20分钟）的存在下进行涡旋。既1和5分钟涡旋没有引起任何明显的组织破坏，而20分钟涡旋导致损害的小区域，类似引起的用作阳性对照（ 补充图1）针针损坏。而表皮或不角质层损伤其它组织的内部破坏，不能排除，这种情况下，似乎相当不可能如1分钟，5分钟处理过的幼虫重新附着在预期图案与时间帧血细胞，提示幼虫完整性不妥协D（ 参考图1）。与此相反，幼虫涡旋20分钟从缺乏再附着遭遇，并没有显示出甚至附着循环血细胞表皮伤口位点，如先前已描述的14。 最后，以证明该方法的再现性，我们比较了2.5毫米幼虫从以上两个实验，这是由不同的实验者进行生物学重复。所示补充图2中，两个队列表明可比总数每幼虫血细胞，和循环血细胞的百分比。学生t检验显示，无统计学差异显著，这表明该方法是可重复的和广泛适用的。 <img alt="图1" src="/files/ftp_upload/52733/52733fig1highres.jpg" width="500" /> 图1.血细胞出血/擦伤和干扰分析小号<html> etup和原理图。 （一）单图片幻灯片设置:五2毫米正方形成像与5倍的目标（B）磁砖扫描幻灯片设置:四3mm见方的成像出血/中≤2.5毫米幼虫瓷砖扫描显微镜擦伤。用于成像的推荐目标是5X或10X。（C）的渗出/使用ImageJ刮测定示意和所得量词。（D）在干扰测定，所述血球图案被机械地通过涡旋幼虫用玻璃珠破碎。幼虫被允许恢复历时45分钟在此期间，血细胞重新粘附到造血口袋。血细胞的粘附特性可以通过这种方法进行评估，量化血细胞中循环障碍后的百分比。 <a href="https://www.jove.com/files/ftp_upload/52733/52733fig1highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 方法"&gt; <img alt="图2" src="/files/ftp_upload/52733/52733fig2highres.jpg" width="700" /> 图2.出血/刮测定释放循环和居民血细胞。 （A）流血幼虫，腹部切口在幼虫的后部和前部末端均采用（剪刀符号）。（二）血细胞在流通将流出切口，解决了滑动表面上。（C）淋巴腺（LG）位于和牵制，没有刺破它。居民血细胞释放被戳和/或刮用针幼虫。幻灯片（四）居民血细胞（E，F）反复刮过程，直到所有常住血细胞释放。包含完整的淋巴腺的幼虫尸体被抛在后面。 <a href="https://www.jove.com/files/ftp_upload/52733/52733fig2highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 图3.自动使用ImageJ血细胞的定量。 （A，B）中的ImageJ打开血球图像文件后， 下限阈值电平被调整，以考虑所有的细胞的图像中。（C，D）分析 颗粒需要设定该单元像素尺寸，圆度，且结果读出格式 （例如，覆盖纲要 ）。（E）摘要窗口显示血细胞的数量。 <a href="https://www.jove.com/files/ftp_upload/52733/52733fig3highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/52733/52733fig4highres.jpg" width="700" /> 图4。代表结果（1）。血细胞数量和居民状态经过了<html>当然幼虫发育。 （A）中的幼虫阶段的概述; 1日龄（48小时AEL;约1.2毫米的长度）; 第二届龄（80小时AEL 2.5毫米的长度）; 3 路龄 （96小时AEL;〜3.5级毫米的长度）。基因型是HMLΔ-GAL4，UAS-GFP;他-GAL4。阶段被评估幼虫mouthhooks证实，在各自的幼虫阶段循环和居民血细胞数（B）条形图。循环血细胞（C）的百分比。注意，循环血细胞的分数超过幼虫发育的过程中增加不相称。（D）的总血细胞，从每幼虫循环和驻地血细胞的总和所得。使用血细胞渗出/刮法进行量化; ñ≥6幼虫/条件，误差线显示标准差，结果在3个独立的重复实验证实。JPG"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图5" src="/files/ftp_upload/52733/52733fig5highres.jpg" width="700" /> 图5.代表性的成果（2）。对影响血细胞居住的机械干扰。 （AC）之前和用玻璃珠，随后45分钟恢复涡旋后幼虫的实施例（A）的无干扰控制。血细胞在玻璃珠和水中的悬浮液涡旋幼虫后定位于造血口袋（B）的血细胞打乱模式，在0 分钟（℃）在恢复后的干扰45分钟血球图案。;许多血细胞已经搬迁到造血的口袋里。注意放大背血管相关的集群和腕背横纹这是早期干扰后的积累（箭头）为主的网站。基因型是HMLΔ-GAL4，UAS-GFP;他-GAL4 x YW。循环血细胞的出血/刮法进行定量的（D）的百分比。（E）的总血细胞，每幼虫流通和居民血细胞的总和产生的。 ñ≥4幼虫/条件，误差线显示标准差，结果在3个独立的重复实验证实。学生的t检验，以确认意义，NS（不显著），**（P≤0.05），**（P≤0.01）。 <a href="https://www.jove.com/files/ftp_upload/52733/52733fig5highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Assaying Blood Cell Populations of the Drosophila melanogaster Larva at JoVE.com

Assaying Blood Cell Populations of the Drosophila melanogaster Larva

Drosophila blood cells, or hemocytes, cycle between resident sites and circulation. In the larva, resident (sessile) hemocytes localize to inductive microenvironments, the Hematopoietic Pockets, while circulating hemocytes move freely in the hemolymph. The goal of this protocol is the standardized isolation and quantification of these two, behaviorally distinct but interchanging, hemocyte populations.

的测定血细胞种群<em&gt;果蝇</em&gt;幼虫

In vertebrates, hematopoiesis is regulated by inductive microenvironments (niches). Likewise, in the invertebrate model organism&#160;Drosophila ...

assaying-blood-cell-populations-of-the-drosophila-melanogaster-larva

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

Assaying Blood Cell Populations of the Drosophila melanogaster Larva

12.3K 次观看.  University of California San Francisco. Drosophila blood cells, or hemocytes, cycle between resident sites and circulation. In the larva, resident (sessile) hemocytes localize to inductive microenvironments, the Hematopoietic Pockets, while circulating hemocytes move freely in the hemolymph. The goal of this protocol is the standardized isolation and quantification of these two, behaviorally distinct but interchanging, hemocyte populations.

视频: Assaying Blood Cell Populations of the Drosophila melanogaster Larva

Methods to Examine the Lymph Gland and Hemocytes in Drosophila Larvae

Many parallels exist between the Drosophila and mammalian hematopoietic systems, even though Drosophila lack the lymphoid lineage that characterize mammalian adaptive immunity. Drosophila and mammalian hematopoiesis occur in spatially and temporally distinct phases to produce several blood cell lineages. Both systems maintain reservoirs of blood cell progenitors with which to expand or replace mature lineages. The hematopoietic system allows Drosophila and mammals to respond to and to adapt to immune challenges. Importantly, the transcriptional regulators and signaling pathways that control the generation, maintenance, and function of the hematopoietic system are conserved from flies to mammals. These similarities allow Drosophila to be used to genetically model hematopoietic development and disease.
Here we detail assays to examine the hematopoietic system of Drosophila larvae. In particular, we outline methods to measure blood cell numbers and concentration, visualize a specific mature lineage in vivo, and perform immunohistochemistry on blood cells in circulation and in the hematopoietic organ. These assays can reveal changes in gene expression and cellular processes including signaling, survival, proliferation, and differentiation and can be used to investigate a variety of questions concerning hematopoiesis. Combined with the genetic tools available in Drosophila, these assays can be used to evaluate the hematopoietic system upon defined genetic alterations. While not specifically outlined here, these assays can also be used to examine the effect of environmental alterations, such as infection or diet, on the hematopoietic system.

Methods to Examine the Lymph Gland and Hemocytes in Drosophila Larvae

Drosophila and mammalian hematopoietic systems share many common features, making Drosophila an attractive genetic model to study hematopoiesis. Here we demonstrate dissection and mounting of the major larval hematopoietic organ for immunohistochemistry. We also describe methods to assay various larval hematopoietic compartments including circulating hemocytes and sessile crystal cells.

方法来检查淋巴腺和血细胞中<em&gt;果蝇</em&gt;幼虫

Many parallels exist between the Drosophila and mammalian hematopoietic systems, even though Drosophila lack the lymphoid lineage that characterize ...

科研

发育生物学

Functional Analysis of the Larval Feeding Circuit in Drosophila

The serotonergic feeding circuit in Drosophila melanogaster larvae can be used to investigate neuronal substrates of critical importance during the development of the circuit. Using the functional output of the circuit, feeding, changes in the neuronal architecture of the stomatogastric system can be visualized. Feeding behavior can be recorded by observing the rate of retraction of the mouth hooks, which receive innervation from the brain. Locomotor behavior is used as a physiological control for feeding, since larvae use their mouth hooks to traverse across an agar substrate. Changes in feeding behavior can be correlated with the axonal architecture of the neurites innervating the gut. Using immunohistochemistry it is possible to visualize and quantitate these changes. Improper handling of the larvae during behavior paradigms can alter data as they are very sensitive to manipulations. Proper imaging of the neurite architecture innervating the gut is critical for precise quantitation of number and size of varicosities as well as the extent of branch nodes. Analysis of most circuits allow only for visualization of neurite architecture or behavioral effects; however, this model allows one to correlate the functional output of the circuit with the impairments in neuronal architecture.

Functional Analysis of the Larval Feeding Circuit in Drosophila

The feeding circuit in Drosophila melanogaster larvae serves a simple yet powerful model that allows changes in feeding rate to be correlated with alterations in the stomatogastric neural circuitry. This circuit is composed of central serotonergic neurons that send projections to the mouth hooks as well as the foregut.

在幼虫馈电电路的功能分析果蝇</i

The serotonergic  feeding circuit in Drosophila  melanogaster larvae can be used to investigate neuronal substrates of  critical importance during the ...

神经科学

A Direct and Simple Method to Assess Drosophila melanogaster's Viability from Embryo to Adult

In Drosophila melanogaster, viability assays are used to determine the fitness of certain genetic backgrounds. Allelic variations can result in partial or complete loss of viability at different stages of development. Our lab has developed a method to assess viability in Drosophila from embryo to fully mature adult. The method relies on quantifying the number of progeny present at different stages during development, starting with hatched embryos. After embryos have been quantified, additional stages are counted, including L1/L2, pupae, and mature adults. After all stages have been examined, a statistical analysis such as the chi-square test is used to determine if there is a significant difference between the starting number of progeny (hatched embryos) and later stages culminating in the observed number of adults, thus rejecting or accepting the null hypothesis (that the number of hatched embryos will be equal to the number of larvae, pupae, and adults recorded throughout the stages of development). The primary advantage of this assay is its simplicity and accuracy, as it does not require an embryo rinse to transfer them to the food vial, avoiding losses from technical errors. Although the protocol described here does not directly examine L2/L3 larvae, additional steps can be added to account for these. Comparing the number of hatched embryos, L1, pupae, and adults can help determine if viability was compromised during the L2/L3 stages for further studies (the use of colored food helps with visual identification of larvae). Overall, this method can help Drosophila researchers and educators determine when viability is compromised during the fly life cycle. Routine assessment of stocks using this assay can prevent accumulation of secondary mutations that may affect the phenotype of the originally isolated mutant, especially if the original mutations affect fitness. For this reason, our lab maintains multiple copies of each of our Dm ime4 alleles and routinely checks the purity of each stock with this method in addition to other molecular analyses.&#160;

A Direct and Simple Method to Assess Drosophila melanogaster's Viability from Embryo to Adult

This protocol is designed to assess the viability of Drosophila at every developmental stage, from embryo to adult. The method can be used to determine and compare the viability of different genotypes or growth conditions.

一种直接而简单的方法，用于评估从胚胎到成人的果蝇黑色素的生存能力

In Drosophila melanogaster, viability assays are used to determine the fitness of certain genetic backgrounds. Allelic variations can result in partial or ...

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we present a protocol for the mounting of Drosophila melanogaster embryos and subsequent live imaging of fluorescently labeled hemocytes, the embryonic macrophages of this organism. Using the Gal4-uas system1 we drive the expression of a variety of genetically encoded, fluorescently tagged markers in hemocytes to follow their developmental dispersal throughout the embryo. Following collection of embryos at the desired stage of development, the outer chorion is removed and the embryos are then mounted in halocarbon oil between a hydrophobic, gas-permeable membrane and a glass coverslip for live imaging. In addition to gross migratory parameters such as speed and directionality, higher resolution imaging coupled with the use of fluorescent reporters of F-actin and microtubules can provide more detailed information concerning the dynamics of these cytoskeletal components.

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Drosophila hemocytes disperse over the entirety of the developing embryo. This protocol demonstrates how to mount and image these migrations using embryos with fluorescently labelled hemocytes.

实时成像果蝇胚胎血细胞迁移

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we ...

生物学

Assay for Blood-brain Barrier Integrity in Drosophila melanogaster

Proper nervous system development includes the formation of the blood-brain barrier, the diffusion barrier that tightly regulates access to the nervous system and protects neural tissue from toxins and pathogens. Defects in the formation of this barrier have been correlated with neuropathies, and the breakdown of this barrier has been observed in many neurodegenerative diseases. Therefore, it is critical to identify the genes that regulate the formation and maintenance of the blood-brain barrier to identify potential therapeutic targets. In order to understand the exact roles these genes play in neural development, it is necessary to assay the effects of altered gene expression on the integrity of the blood-brain barrier. Many of the molecules that function in the establishment of the blood-brain barrier have been found to be conserved across eukaryotic species, including the fruit fly, Drosophila melanogaster. Fruit flies have proven to be an excellent model system for examining the molecular mechanisms regulating nervous system development and function. This protocol describes a step-by-step procedure to assay for blood-brain barrier integrity during the embryonic and larval stages of D. melanogaster development.

Assay for Blood-brain Barrier Integrity in Drosophila melanogaster

Blood-brain barrier integrity is critical for nervous system function. In Drosophila melanogaster, the blood-brain barrier is formed by glial cells during late embryogenesis. This protocol describes methods to assay for blood-brain barrier formation and maintenance in D. melanogaster embryos and third instar larvae.

果蝇黑色素血脑屏障完整性分析

Proper nervous system development includes the formation of the blood-brain barrier, the diffusion barrier that tightly regulates access to the nervous ...

Extraction of Hemocytes from Drosophila melanogaster Larvae for Microbial Infection and Analysis

During the pathogenic infection of Drosophila melanogaster, hemocytes play an important role in the immune response throughout the infection. Thus, the goal of this protocol is to develop a method to visualize the pathogen invasion in a specific immune compartment of flies, namely hemocytes. Using the method presented here, up to 3 &#215; 106 live hemocytes can be obtained from 200 Drosophila 3rd instar larvae in 30 min for ex vivo infection. Alternatively, hemocytes can be infected in vivo through injection of 3rd instar larvae followed by hemocyte extraction up to 24 h post-infection. These infected primary cells were fixed, stained, and imaged using confocal microscopy. Then, 3D representations were generated from the images to definitively show pathogen invasion. Additionally, high-quality RNA for qRT-PCR can be obtained for the detection of pathogen mRNA following infection, and sufficient protein can be extracted from these cells for Western blot analysis. Taken together, we present a method for definite reconciliation of pathogen invasion and confirmation of infection using bacterial and viral pathogen types and an efficient method for hemocyte extraction to obtain enough live hemocytes from Drosophila larvae for ex vivo and in vivo infection experiments.

Extraction of Hemocytes from Drosophila melanogaster Larvae for Microbial Infection and Analysis

This method demonstrates how to visualize pathogen invasion into insect cells with three-dimensional (3D) models. Hemocytes from Drosophila larvae were infected with viral or bacterial pathogens, either ex vivo or in vivo. Infected hemocytes were then fixed and stained for imaging with a confocal microscope and subsequent 3D cellular reconstruction.

从果蝇幼虫中提取包囊微生物感染及分析

During the pathogenic infection of Drosophila melanogaster, hemocytes play an important role in the immune response throughout the infection. Thus, the ...

免疫与感染

Assessing the Cellular Immune Response of the Fruit Fly, Drosophila melanogaster, Using an In Vivo Phagocytosis Assay

In all animals, innate immunity provides an immediate and robust defense against a broad spectrum of pathogens. Humoral and cellular immune responses are the main branches of innate immunity, and many of the factors regulating these responses are evolutionarily conserved between invertebrates and mammals. Phagocytosis, the central component of cellular innate immunity, is carried out by specialized blood cells of the immune system. The fruit fly, Drosophila melanogaster, has emerged as a powerful genetic model to investigate the molecular mechanisms and physiological impacts of phagocytosis in whole animals. Here we demonstrate an injection-based in vivo phagocytosis assay to quantify the particle uptake and destruction by Drosophila blood cells, hemocytes. The procedure allows researchers to precisely control the particle concentration and dose, making it possible to obtain highly reproducible results in a short amount of time. The experiment is quantitative, easy to perform, and can be applied to screen for host factors that influence pathogen recognition, uptake, and clearance.

Assessing the Cellular Immune Response of the Fruit Fly, Drosophila melanogaster, Using an In Vivo Phagocytosis Assay

This protocol describes an in vivo phagocytosis assay in adult Drosophila melanogaster to quantify phagocyte recognition and clearance of microbial infections.

果蝇、果蝇细胞免疫应答的评估

In all animals, innate immunity provides an immediate and robust defense against a broad spectrum of pathogens. Humoral and cellular immune responses are ...

Live Cell Cycle Analysis of Drosophila Tissues using the Attune Acoustic Focusing Cytometer and Vybrant DyeCycle Violet DNA Stain

Flow cytometry has been widely used to obtain information about DNA content in a population of cells, to infer relative percentages in different cell cycle phases. This technique has been successfully extended to the mitotic tissues of the model organism Drosophila melanogaster for genetic studies of cell cycle regulation in vivo. When coupled with cell-type specific fluorescent protein expression and genetic manipulations, one can obtain detailed information about effects on cell number, cell size and cell cycle phasing in vivo. However this live-cell method has relied on the use of the cell permeable Hoechst 33342 DNA-intercalating dye, limiting users to flow cytometers equipped with a UV laser. We have modified this protocol to use a newer live-cell DNA dye, Vybrant DyeCycle Violet, compatible with the more common violet 405nm laser. The protocol presented here allows for efficient cell cycle analysis coupled with cell type, relative cell size and cell number information, in a variety of Drosophila tissues. This protocol extends the useful cell cycle analysis technique for live Drosophila tissues to a small benchtop analyzer, the Attune Acoustic Focusing Cytometer, which can be run and maintained on a single-lab scale.

Live Cell Cycle Analysis of Drosophila Tissues using the Attune Acoustic Focusing Cytometer and Vybrant DyeCycle Violet DNA Stain

A protocol for cell cycle analysis of live Drosophila tissues using the Attune Acoustic Focusing Cytometer is described. This protocol simultaneously provides information about relative cell size, cell number, DNA content and cell type via lineage tracing or tissue specific expression of fluorescent proteins in vivo.

活细胞周期分析<em&gt;果蝇</em&gt;组织使用同调声聚焦流式细胞仪Vybrant的DyeCycle紫DNA染色

Flow cytometry has  been widely used to obtain information about DNA content in a population of  cells, to infer relative percentages in different cell ...

Assessing the Age-Specific Phagocytic Ability of Adult Drosophila melanogaster Hemocytes using an In Vivo Phagocytosis Assay

Phagocytosis is an essential function of the innate immune response. This process is carried out by phagocytic hemocytes whose primary function is to recognize a wide range of particles and destroy microbial pathogens. As organisms age, this process begins to decline, yet little is known about the underlying mechanisms or the genetic basis of immunosenescence. Here, an injection based in vivo phagocytosis assay is used to assess age related changes in different aspects of phagocytosis, such as binding, engulfment, and degradation of internalized particles, by quantifying phagocytic events in hemocytes in adult Drosophila. Drosophila melanogaster has become an ideal model to investigate age related changes in innate immune function for many reasons. For one, many genetic components and functions of the innate immune response, including phagocytosis, are evolutionarily conserved between Drosophila and mammals. Because of that, results obtained from using this protocol are likely to be widely relevant to understanding the age related changes in immune function in a variety of organisms. Additionally, we note that this method provides quantitative estimates of hemocyte phagocytic ability, which could be useful for a variety of research topics, and need not be limited to studies of aging.

Assessing the Age-Specific Phagocytic Ability of Adult Drosophila melanogaster Hemocytes using an In Vivo Phagocytosis Assay

This protocol describes an in vivo assay of phagocytosis used to assess and quantify the ability of young and aged Drosophila melanogaster hemocytes to phagocytose bacteria.

使用 Vivo 法戈西西亚赛评估成人嗜血杆菌黑色粒细胞的年龄特异性噬细胞能力

Phagocytosis is an essential function of the innate immune response. This process is carried out by phagocytic hemocytes whose primary function is to ...

Cuticle Disruption: A Method to Collect Hemolymph from Drosophila Larvae

Source: Reimels, T. A. and Pfleger, C. M. <a href="https://www.jove.com/video/54544/" target="_blank">Methods to Examine the Lymph Gland and Hemocytes in Drosophila Larvae</a>. J. Vis. Exp. (2016).
The Drosophila hemolymph serves as both blood and interstitial fluid, circulating throughout the body during the larva and adult stages. This video describes a protocol to collect larval hemolymph in order to quantify the circulating cells.

角质层破坏:一种从果蝇幼虫中收集血淋巴的方法

Source: Reimels, T. A. and Pfleger, C. M. Methods to Examine the Lymph Gland and Hemocytes in Drosophila Larvae. J. Vis. Exp. (2016).
The Drosophila ...

的测定血细胞种群

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方法来检查淋巴腺和血细胞中

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